IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/08/10

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August 10, 2010

I was saddened to see that none of my transformations worked!! I went back and ran a gel of all of my digests to see what was going wrong. Here is the picture:

I found that one of my most important parts; the B0015 terminator, was not showing up on the gel. I decided to use Erin's B0015 for my third attempt at transforming these genes. So I did a lone digestion of her B0015.

Digestion of B0015 (labeled as RXN 0)-

  • 2.5 uL B0015
  • 39.0 uL dH2O
  • 5 uL NEBuffer 4
  • 0.5 uL BSA
  • 1.0 uL XbaI
  • 2.0 uL PstI

I let the digest incubate in the 37 C bath for 15 minutes, then put it into the80 C bath for 20 minutes.

I then retried yesterdays ligations using the new B0015. All of these reactions are from 8/9/10 <-crazy!

Ligations
1 2 3 4 5
11 uL dH2O 11 uL dH2O 11 uL dH2O 11 uL dH2O 11 uL dH2O
2.0 uL 10X T4 DNA Ligase buffer 2.0 uL 10X T4 DNA Ligase buffer 2.0 uL 10X T4 DNA Ligase buffer 2.0 uL 10X T4 DNA Ligase buffer 2.0 uL 10X T4 DNA Ligase buffer
1.0 uL T4 DNA ligase 1.0 uL T4 DNA ligase 1.0 uL T4 DNA ligase 1.0 uL T4 DNA ligase 1.0 uL T4 DNA ligase
2.0 uL Digest 1 2.0 uL Digest 0 2.0 uL Digest 1 (From 7/29 (J23100)) 2.0 uL Digest 5 (From 7/29 (C0012)) 2.0 uL Digest 4
2.0 uL Digest 0 2.0 uL Digest 3 2.0 uL Digest 5 2.0 uL Digest 0 2.0 uL Digest 0
2.0 uL Digest 7 2.0 uL Digest 7 2.0 uL Digest 7 2.0 uL Digest 7 2.0 uL Digest 7

This time I used heat shock to transform cells. I used 50 uL of the heat-shock competent cells and 2 uL of my ligation. I put the mixture in 2.5 mL centrifuge tubes and put o nice for 10 minutes. Then put in 42 C water bath fro 45 seconds, and back on ice for 2 minutes. Then I added 500 uL of SOC media to the tubes and put in the 37 C room for 1 hour to recover. After I took those out they were plated on CAP plates and put into the 37 C room for overnight.



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